The present invention relates to compositions and methods of use for a novel putative GTPase activating protein (xe2x80x9cGAPxe2x80x9d) that interacts with papilloma virus E6 oncoprotein and is thereby targeted for degradation. Said novel protein, herein designated xe2x80x9cE6TP1xe2x80x9d for E6 targeted protein, its nucleic acid and polypeptide sequences, and derivatives, fragments, and analogues thereof, are described. The invention further relates to cells containing recombinant E6TP1 nucleic acid and protein sequences; animal models of the same, including transgenic and xe2x80x9cknock-outxe2x80x9d animal models; anti-E6TP1 specific antibodies, and derivatives, fragments, and analogues thereof; oligonucleotides hybridizable to sense or antisense E6TP1 nucleic acids; oligonucleotide pairs capable of PCR amplifying E6TP1; and use of any of the above compositions in diagnostics, therapeutics, and treatments of tumors. Specific embodiments include methods of use of E6TP1 in tumor suppression. In addition, the present invention relates to compositions and methods of use of E6TP1 in high-risk human papilloma virus (xe2x80x9cHPVxe2x80x9d) associated carcinomas of cervix and other anogenital tumors.
Carcinomas, the tumors originating from epithelial cells, constitute nearly 80% of all human cancers. In the U.S. alone, a predicted 650,000 new cases of the carcinomas of lung, colon, breast, prostate and cervix will be diagnosed in 1998, and nearly 300,000 of these will be fatal. See, e.g., American Cancer Society Cancer Facts and Figures, American Cancer Society, Inc. (1998). Despite these astonishing statistics, much of our knowledge into cell biology of the oncogenic process stems from studies of fibroblasts, reflecting the available experimental models.
A critical event in carcinogenesis is the conversion of normal epithelial cells, with a finite proliferative potential, into cells that are endowed with an ability to multiply continuously, a trait that allows accumulation of further genetic alterations enroute to full malignancy. In vitro, this behavior manifests as continuous proliferation of cells beyond their limited life span. This process is referred to as immortalization. Understanding the biochemical basis of immortalization is therefore likely to identify crucial cellular pathways involved in cellular control. The recently identified ability of viral oncogenes to immortalize normally senescent epithelial cells has provided practical approaches to delineate the regulatory cascades involved in these critical paths.
For example, normal human mammoplasty-derived mammary epithelial cells reproducibly proliferate in vitro for about 20 passages, followed by their senescence. See, e.g., Band and Sager, Proc Natl Acad Sci USA., 86:1249-1253 (1989); Band, Sem Cancer Biol, 6: 185-192 (1995). A similar finite life span is exhibited in vitro by other epithelial cells. A number of viral oncogenes, including SV40 large T and adenovirus E1A and E1B, were inefficient at immortalizing the epithelial cells, in sharp contrast to rodent fibroblasts which were efficiently transformed by these oncoprotein. See, e.g., Sager, Cancer Cells, 2: 487-494 (1984). Strikingly, however, we found that transfection of the genome of high risk human papilloma viruses (xe2x80x9cHPVsxe2x80x9d)-16 or -18, which are naturally associated with epithelial oncogenesis in the genitourinary tract, led to reproducible and highly efficient immortalization of MECs. See, e.g., Band et al., Proc Natl Acad Sci USA, 87: 463-467 (1990). The HPV genome transfection also efficiently immortalized keratinocytes (a natural host epithelial cell type for these viruses) and other epithelial cell types. See, e.g., Kaur and McDougall, J Virol, 62: 1917-1924 (1988); Yeager et al., Cancer Res, 55: 493-497 (1995). In all of these cases, the HPV genome-transformed cells were immortal but not fully tumorigenic, as shown by their inability to grow in soft agar or form tumors when implanted in nude mice. However, when these immortal cells are transfected with additional oncogenes (e.g., activated H-ras or mutant erbB-2), they attained a tumorigenic phenotype. See, e.g., Woodworth, In: Neoplastic Transformation in Human Cell Culture: Mechanisms of Carcinogenesis, Eds. Rhim and Dritschilo, Humana Press, New Jersey, pp 153-161 (1991). Thus, introduction of the HPV genome into primary human epithelial cells induces a preneoplastic transformation.
The human papilloma viruses (xe2x80x9cHPVsxe2x80x9d) are associated with epithelial tumors or benign lesions, especially those of anogenital origin. See, e.g., Zur Hausen and Schneider, xe2x80x9cThe Papillomavirusesxe2x80x9d In: The Papillomaviruses, Howley and Salzman (ed.), Vol. 2 of The Papovaviridae, (Plenum, New York) pp 245-263 (1987). These viruses are grouped into low-risk HPVs, such as HPV6 and HPV11, which are usually associated with benign warts, and high-risk HPVs, such as HPV 16 and HPV18, which are associated with carcinomas of cervix and other genital tumors. See, e.g., Zur Hausen, ibid. Previous studies have identified two viral oncoproteins xe2x80x9cE6xe2x80x9d and xe2x80x9cE7xe2x80x9d, which are expressed in the majority of HPV-associated carcinomas, and which functionally inactivate cellular tumor suppressor proteins p53 and retinoblastoma (Rb), respectively. See, e.g., Dyson et al., Science 243: 934-937 (1989); Huibregtse et al., Mol Cell Biol 13: 4918-4927 (1993); Scheffner et al., Cell 75: 495-505 (1993). This is thought to provide the basis for the ability of high-risk, but not the low-risk, HPVs to promote oncogenesis.
In recent years, a distinct mechanism of viral oncoprotein-induced inactivation of tumor suppressor protein function has emerged, involving the targeting of tumor suppressor protein(s) to ubiquitin-proteasome mediated degradation machinery. See, e.g., Boyer et al., ibid.; Ciechanover, Cell 79: 13-21 (1994); Huibregtse et al., Mol Cell Biol 13: 4918-4927 (1993). The ubiquitin-proteasome pathway participates in physiological regulation of the levels of cell-cycle related proteins such as cyclins, cdks, and tumor suppressor proteins such as p53 and Rb proteins. See, e.g., Boyer et al., ibid.; Ciechanover, ibid.; Maki et al., Cancer Res 56: 2649-2654 (1996). Viral oncoproteins target cellular tumor suppressor proteins to this pathway for enhanced degradation. See, e.g., Huibregtse et al., EMBO J 10: 4129-4135 (1991); Scheffner et al., Cell 63: 1129-1136 (1990). High-risk HPV E6 oncoproteins associate with E6-AP, a ubiquitin ligase, which in turn interacts with p53 and targets it for degradation. See, e.g., Huibregtse et al (1991), ibid.; Scheffner et al., ibid. We have since found that the high-risk HPV16 E7 oncoprotein also uses the ubiquitin proteasome pathway for enhanced degradation of bound Rb protein. See, e.g, Boyer et al., Cancer Res 56: 4620-4624 (1996).
Characterization of oncogenesis-related cellular targets of HPV oncoproteins has been facilitated by the ability of HPV to dominantly immortalize primary human cells in vitro. See, e.g., supra; Band et al., Proc Natl Acad Sci USA 87: 463-467 (1990); Woodworth et al., J Virol 63: 159-164 (1989). Both E7 and E6 proteins of high-risk HPVs are required for efficient immortalization of cervical keratinocytes, E6 alone is not enough. See, e.g., Hawley-Nelson et al., EMBO J 8: 3905-3910 (1989); Munger et al., J Virol 63: 4417-4421 (1989).
Surprisingly, we found that HPV E6 alone is sufficient to immortalize a subtype of normal human mammary epithelial cells (xe2x80x9cMECsxe2x80x9d). See, e.g., Band et al., J Virol 65: 6671-6676 (1991). Use of HPV16 DNA constructs with disruption of individual open reading frames of early (xe2x80x9cExe2x80x9d) viral genes demonstrated that E1, E2, E4 and E7 genes were dispensable for MEC immortalization. In contrast, the E6 open reading frame was indispensable. See, e.g., Band et al., ibid. Introduction of the E6 gene alone demonstrated that this gene was sufficient to immortalize MECs. See, e.g., Band et al., EMBO J, 12: 1847-1852 (1993). Thus, immortalization of the human MECs by E6 provides a simple single-oncogene model wherein to define the cellular pathways whose alterations underlie immortalization of epithelial cells. See e.g., Band et al., ibid.
Mutational analysis of HPV16 E6 revealed a direct correlation between MEC immortalization and the ability of E6 proteins to induce in vivo p53 degradation in these cells, confirming the crucial role enhanced p53 degradation plays in E6-mediated immortalization. See, e.g., Band et. al., EMBO J 12: 1847-1852, (1993); Dalal et al., J Virol 70: 683-688 (1996). However, when tested, loss of p53 itself was insufficient for immortalization. Of a panel of dominant-negative p53 missense mutants, known to complex with and functionally inactivate the endogenous wild-type p53 protein, nine out of the twelve mutants tested failed to immortalize MECs, even though they were expressed at high levels. See, e.g., Scheffner et al., J Virol 66: 5100-5105 (1992); Takahashi et al. Science, 246: 491-494 (1989). Furthermore, the remaining three mutants immortalized the MECs much less efficiently compared to HPV16 E6. See, e.g., Band et al., EMBO J, 12: 1847-1852 (1993); Dalal et al., J Virol, 70: 683-688 (1996); Gao et al., Cancer Res, 56: 3129-3133 (1996); and Cao et al., Cancer Res, 57: 5584-5589(1997). Together, these results strongly suggest that the HPV 16 E6 oncoprotein targets additional biochemical pathways whose functional inactivation is important for immortalization.
Consistent with this possibility, HPV16 E6 has recently been shown to interact with three proteins: xe2x80x9cERC55xe2x80x9d, a putative calcium binding protein; xe2x80x9cpaxillinxe2x80x9d, a protein involved in transducing signals from the plasma membrane to the actin cytoskeleton; and xe2x80x9chDlgxe2x80x9d, the human homologue of the Drosophila discs large tumor suppressor protein. See, e.g., Chen et al., Science 269: 529-531 (1995); Kiyono et al., Proc Natl Acad Sci USA 94: 11612-11616 (1997); Lee et al., Proc Natl Acad Sci USA 94: 6670-6675 (1997); Tong and Howley, Proc Natl Acad Sci USA 94: 4412-4417 (1997). Binding of these proteins to high or low risk HPV E6 mutants correlates with the immortalizing ability of the E6 proteins, suggesting a potential role for these non-p53 E6-binding proteins in cellular transformation, although direct studies to demonstrate such a role have not been carried out.
In nonviral associated carcinomas, when the gene that encodes tuberin (xe2x80x9cTSC2xe2x80x9d), a tumor suppressor protein frequently mutated in the autosomal dominant syndrome of tuberous sclerosis, was introduced into a renal carcinoma cell line derived from the Eker rat, a model of hereditary renal carcinoma, tumorigenicity was suppressed. See, e.g., Jin et al., Proc Natl Acad Sci USA 93: 9154-9159 (1996).
Signals that activate growth, and therefore antagonize the function of cellular tumor suppressor proteins, are mediated through growth factor receptors located on the plasma membrane that interact with highly regulated signaling pathways. The small Ras-like GTPases are a family of signaling proteins that includes Ras, Rap1, Rap2, R-ras, TC21, Ral, and Rhob, and are defined by a common homology domain known in Ras to interact with downstream targets. Mutant Ras proteins are found in around 15% of all human tumors, and in 50 to 90% of all adenocarcinomas of the colon and pancreas, respectively. See, e.g., Bos, EMBO J 17: 6776-6782 (1998). Therefore, the Ras family of proteins plays an important role in non-HPV associated carcinomas.
Ras and Ras-related proteins act as molecular switches for controlling cellular signaling pathways by cycling between GTP-bound xe2x80x9conxe2x80x9d and GDP-bound xe2x80x9coffxe2x80x9d states. In the GTP-bound xe2x80x9conxe2x80x9d state, these proteins interact with and activate effector proteins (e.g., raf activation by Ras). Intrinsic GTPase activity hydrolyzes GTP to GDP, returning the G-binding proteins to the xe2x80x9coffxe2x80x9d (GDP-bound) state. Two families of regulatory proteins control the G-protein cycle. Guanine nucleotide exchange factors (GEFs) stimulate the release of bound GDP, promoting GTP loading. See, e.g., Feig, Curr Opin Cell Biol, 6: 204-211 (1994); Polakis and McCormick, Cancer Surveys, 12: 25-42 (1992); Polakis and McCormick, J Biol Chem, 268: 9157-9160 (1993). In contrast, GTPase-activating proteins (GAPs) stimulate the otherwise slow intrinsic GTPase activity by several orders of magnitude, inducing GTP hydrolysis to GDP and returning the G-binding proteins to an xe2x80x9coffxe2x80x9d state. Many members of this protein signaling family act as activated oncogenes in their mutated form. Oncogenic mutations of Ras invariably lead to an inability of GAP proteins to stimulate GTPase activity, resulting in a constitutively xe2x80x9conxe2x80x9d state. Thus, GAP proteins provide a critical regulatory mechanism to ensure normal function of small G proteins.
Analogous to the Ras-specific GEF xe2x80x9cson-of-sevenlessxe2x80x9d (xe2x80x9cSOSxe2x80x9d), a Rap-specific GEF xe2x80x9cC3Gxe2x80x9d has been recently identified. See, e.g., Ichiba et al., J. Biol Chem., 35: 22215-22220 (1997); Okada et al., EMBO J, 17: 2554-2565 (1998). C3G associates with the SH3 domains of the Crk adaptor proteins and their SH2 domains recruit it to tyrosine phosphorylated membrane receptors near the membrane-associated Rap (e.g., epidermal growth factor receptor (EGFR) and nerve growth factor receptor (NGFR). See, e.g., Okada and Pessin, J. Biol Chem., 272: 28179-28182 (1997); York et al., Nature, 392: 622-626 (1998). However, while a clear role has been delineated of Ras G-protein pathway in controlling cell proliferation and differentiation through MAP kinase cascades, the role of Rap1, a known suppressor of Ras activity, has remained enigmatic.
Rap1 was originally identified by its ability to revert the phenotype of viral K-ras transformed NIH3T3 cells. See, e.g., Kitayama et al., Cell 56: 77-84 (1989). Rap1, whose effector domain is nearly identical to that of Ras, was shown to interact with the Ras effectors. See, e.g., Frech et al., Science 249: 169-171 (1990); Hata et al., J Biol Chem 265: 7104-7107 (1990); Herrmann et al., J Biol Chem 271: 6794-6800 (1996). Thus it is hypothesized that Rap1 antagonizes Ras function by sequestering Ras effectors in inactive complexes (e.g., constitutively activate RapV12 mutant inhibited EGF-stimulated Ras-mediated MAP kinase activation in rat fibroblasts). See, e.g., Cook et al., EMBO J, 12: 3475-3485 (1993).
Surprisingly, however, Vossler et al. showed in PC12 cells that Rap1, activated either by mutation or by the cAMP-dependent protein kinase (xe2x80x9cPKAxe2x80x9d), is a selective activator of B-Raf, which activates the MAP kinase cascade and leads to sustained activation of the transcription factor Elk-1. Vossler et al., Cell 89: 73-82 (1997). Also, Yoshida et al. showed that the microinjection of activated Rap1 into Swiss 3T3 cells enhanced the mitogenic signaling pathways in response to insulin. Yoshida et al., Mol Cell Biol 12: 3407-3414 (1992). Transfection of Rap1 into Swiss 3T3 cells increased cell proliferation and oncogenically transformed the cells. See, e.g., Altschuler and Ribeiro-Neto, Proc Natl Acad Sci USA 95: 7475-7479 (1998). In addition, expression in 293 HEK cells of the Rap GEF C3G activated the JNK pathway. See, e.g, Tanaka and Hanafusa, J Biol Chem, 273: 1281-1284 (1998). Rap GTP loading has also been demonstrated upon stimulation through the T cell receptor and thrombin activation of platelets. See, e.g., Reedquist and Bos, J Biol Chem, 9: 4944-4949 (1998); Franke et al., EMBO J, 16: 252-259 (1997). Finally, Rap mainly localizes intracellularly at the endocytic and lysosomal vesicles, whereas Ras localizes to the plasma membrae. See, e.g., Pizon et al., Exp Cell Res 246: 56-68 (1999); Bos EMBO J 23: 6776-6782 (1998). These results suggest that the function of Rap signaling on the Ras pathway is likely to depend on the cell type, the type of receptors, the particular Rap proteins expressed and their localization, and the nature of effector proteins expressed (e.g., Raf vs B-raf).
Thus, a need remains in the art for the identification of tumor suppressors involved in specific carcinomas and in HPV-associated disease states. In addition, identification of cellular factors, other than p53, that are both bound by immortalization-competent HPV E6 and are targeted for enhanced degradation is key to finding a more viable control of tumor suppression and finding potential new therapeutics for treating all stages of HPV-induced disease.
Elucidation of biochemical pathways whose inactivation leads to oncogenic transformation of epithelial cells, the precursors for carcinomas, is a central goal in cancer biology. Normal epithelial cell growth is tightly controlled, and these cells live for a finite life span before they senesce and die. An essential initial step in tumorigenesis involves loss of senescence, or immortalization, which allows a cell to grow indefinitely and to go through further oncogenic steps resulting in fully malignant behavior. We have demonstrated that primary human mammary epithelial cells (xe2x80x9cMECsxe2x80x9d) are efficiently immortalized by a single oncogene, namely HPV16 E6, providing a simple model to elucidate mechanisms of cellular transformation uncomplicated by the effects of additional oncogenes required for efficient immortalization of other epithelial cells.
We used a modified yeast two-hybrid system (see, e.g., Chien et al., Proc Natl Acad Sci USA 88: 9578-9582 (1991)) to identify a novel putative GAP protein, xe2x80x9cE6TP1xe2x80x9d (for xe2x80x9cE6-Targeted Proteinxe2x80x9d) that interacts with high-risk HPV E6 proteins and is targeted for in vitro and in vivo degradation. The ability of E6TP1 to selectively interact with immortalizing HPV16 E6 mutants, but not non-immortalizing HPV16 E6 mutants, implicates this protein in E6-mediated oncogenesis. Two isoforms of human E6TP1 were cloned, namely E6TP1xcex1 and E6TP1xcex2. Therefore, the present invention relates to full length E6TP1xcex1 complementary DNA (xe2x80x9ccDNAxe2x80x9d) nucleotide sequence (GenBank Accession Number AF090989) [SEQ ID NO:1]; E6TP1xcex1 predicted amino acid sequence [SEQ ID NO:2]; full length E6TP1xcex2 cDNA nucleotide sequence (GenBank Accession Number AF090990) [SEQ ID NO:3]; and E6TP1xcex2 predicted amino acid sequence [SEQ ID NO:4]; as well as fragments, derivatives, and analogues of said E6TP1xcex1 and E6TP1xcex2 nucleic acid and amino acid sequences. The invention further provides cells containing recombinant E6TP1 nucleic acid and amino acid sequences, or fragments, derivatives, and analogues thereof; recombinant animal models thereof; anti-E6TP1 specific antibodies, and derivatives, fragments, and analogues thereof; oligonucleotides hybridizable to sense or antisense E6TP1 nucleic acids, and use of any of the above in diagnostics, therapeutics, and treatments of tumors, including use of E6TP1 in tumor suppression and prevention of HPV-associated carcinomas. Methods of isolation and purification of E6TP1 protein are also provided.
The E6TP1 protein exhibits high homology to the GTPase-activating proteins (xe2x80x9cGAPsxe2x80x9d) xe2x80x9cSPA-1xe2x80x9d, xe2x80x9ctuberinxe2x80x9d, and xe2x80x9cRap1GAPxe2x80x9d, three GAPs known to regulate the Rap protein. Rap is a Ras-like GTPase localized mainly near endocytic and lysosomal vesicles in the cell interior. See, e.g., Bos, EMBO J 17: 6776-6782. The mRNA for E6TP1 is widely expressed in tissues and in in vitro cultured cell lines. The gene for E6TP1 localizes to chromosome 14q23.2-14q24.3, within a locus that has been shown to undergo loss of heterozygosity in malignant meningiomas. Importantly, E6TP1 is targeted for degradation by the high-risk, but not the low-risk, HPV E6 proteins both in vitro and in vivo. Furthermore, the immortalization-competent but not the immortalization-incompetent HPV16 E6 mutants target the E6TP1 protein for degradation. This novel target for E6 oncoprotein provides a potential link between HPV E6 oncogenesis and alteration of a small G-protein signaling pathway.
Epithelial cells are the source of all forms of carcinoma. E6TP1 is likely involved in the maintenance of the untransformed state of normal epithelial cells, as we have shown that E6-induced degradation of this protein constitutes one of the lesions identified upon E6-induced cellular immortalization. Therefore, E6TP1 defines a novel biochemical pathway of inhibitory control of cell growth whose function is critical to prevent oncogenic transformation. As noted in the BACKGROUND Section, supra, transfection experiments that replaced the p53 and Rb tumor suppressors in carcinoma cells demonstrated that said replacement of the target protein of HPV oncoprotein was alone sufficient to halt HPV-induced transformation and cell proliferation. Herein we identify E6TP1 as a novel putative tumor suppressor in carcinoma cells. As such, our invention encompasses methods of use of E6TP1 in diagnosis, prevention, and treatment of carcinomas and related diseases.
Various embodiments of the invention include administrating E6TP1 nucleic acids and proteins as a therapeutic agent, regulating E6TP1-associated growth inhibition and tumor suppressor pathways, using E6TP1 in diagnosis of disease states, treating and preventing HPV-associated infections, and using E6TP1-specific antibodies and antisense nucleic acids for modulating E6TP1 and associated proteins and pathways. Also included are methods of administrating E6TP1 in carcinomas and HPV-associated diseases pathway. In addition, the present invention provides compositions and methods of using E6TP1 in high-risk human papilloma virus (xe2x80x9cHPVxe2x80x9d) associated carcinomas of cervix and other anogenital tumors. Methods of identifying E6TP1-associated proteins are provided, in order to provide newer targets for future development of gene-based and other associated diagnostic and therapeutic strategies.